The mechanisms by which eukaryotic organisms regulate gene expression are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. With regard to the process of transcriptional initiation, a wide variety of experiments have pointed to common molecular mechanisms in eukaryotic organisms ranging from humans to yeasts. Previous analysis of the yeast his3 gene has defined DNA sequences necessary for expression and regulation, has identified and characterized GCN4 protein, which is necessary for transcriptional activation, and has shown that expression is governed by two promoters that work by distinct molecular mechanisms. This proposal will investigate several basic issues concerning the molecular mechanisms involved in his3 transcriptional regulation by combining a wide variety of techniques including recombinant DNA technology, molecular and classical yeast genetics, and protein and nucleic acid biochemistry. First, the molecular basis for specific binding of GCN4 protein to the his3 regulatory site will be addressed by using degenerate oligonucleotides to create a large number of mutations within the DNA-binding domain of GCN4. The mutants will be examined in vivo and in vitro for DNA-binding, dimerization, alterations in DNA sequence recognition, and overall structure. Second, the critical features of the short acidic region of GCN4 required for transcriptional activation will be defined by deletion and point mutational analysis. Peptides containing a functional activation region will be synthesized and analyzed for effects on chromatin structure and for potential interactions with other proteins. Third, proteins interacting with GCN4 will be sought by biochemical approaches or by genetic selections to identify the the encoding genes. Biochemical approaches include affinity chromotography to GCN4 or the activation peptide and detection of GCN4 protein-DNA complexes with altered electrophoretic mobilities. Genetic approaches include revertants of gcn4 transcriptional activation mutants and a novel selection scheme involving recombinant DNA libraries in a protein fusion vector. The genes and the encoded proteins will be characterized by standard techniques of yeast molecular biology. Fourth, proteins interacting with either of the two functionally distinct his3 TATA elements will be sought by standard purification or by obtaining suppressor mutations that revert the transcriptional defects of TATA point mutations. The genes encoding these proteins will be cloned with the goal of elucidating their structure and function. Fifth, the molecular basis of constitutive his3 expression will be examined by varying the length and quality of the poly (dA-dT) sequence that serves as the upstream promoter element. The effects on transcription will be correlated with DNA bending and affects on chromatin structure. In summary, the proposed experiments should provide important information on basic questions such as the molecular nature of protein-DNA interactions in eukaryotic gene regulation, interactions between upstream activator proteins and the general transcription machinery, and the relationship of chromatin structure to gene expression.